RESEARCH ARTICLE
Normalized real-time PCR for diagnosis of H. pylori infection Ebtisam E. Al-Moayad1, Saeed M. Alghalibi1, Hassan A. Al-Shamahy2, Akram T. Nasher3, Nezar N. Al-hebshi4,5 ABSTRACT Address for Correspondence: Nezar N. Al-hebshi 1 Department of Biology, Microbiology Section, Faculty of Science, Sana’a University, Yemen 2 Department of Medical Microbiology, Faculty of Medicine and Health Sciences, Sana’a University, Yemen 3 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Sana’a University, Yemen 4 Molecular Biology Research Laboratory, Faculty of Medical Sciences, University of Science and Technology, Sana’a, Yemen 5 Medical Research Center, Jazan University, Jazan, Kingdom of Saudi Arabia Email: [email protected]
http://dx.doi.org/10.5339/qmj.2014.19 Submitted: 16 October 2014 Accepted: 26 November 2014 ª 2014 Al-Moayad, Alghalibi, Al-Shamahy, Nasher, Al-hebshi, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 4.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.
Cite this article as: Al-Moayad EE, Alghalibi SM, Al-Shamahy HA, Nasher AT, Al-hebshi NN. Normalized real-time PCR for diagnosis of H. pylori infection, Qatar Medical Journal 2014:19 http://dx.doi.org/10.5339/ qmj.2014.19
Objectives: There is increasing interest in the use of quantitative PCR (q-PCR) for diagnosis of H. pylori infection. However, the assay remains largely unstandardized, making comparison between studies unreliable. The objective of this study was to assess accuracy of a normalized q-PCR assay for diagnosis of the infection. Subjects and methods: Seventy-six fresh gastric biopsy specimens were collected from patients undergoing upper gastrointestinal tract endoscopy and examined by rapid urease test (RUT), culture, and a commercial TaqMan q-PCR assay targeting the ureA gene. Counts obtained from the latter assay were normalized to the human ACTB gene. A subject was considered to be infected if two or more assays were positive. Results: The detection rates were 42.1%, 52.6%, and 78.9% by culture, RUT and q-PCR, respectively. Bacterial density ranged 0.005 to 4800 bacteria per 100 human cells. Because q-PCR showed low initial specificity (45.7%), the cutoff value for the assay was recalculated as 1 bacterium per 100 human cells, using ROC curve analysis. Accordingly, the sensitivities and specificities were 79.5% and 97.3%, respectively, for culture; 94.9% and 91.9%, respectively, for RUT; and 94.9% and 94.6%, respectively, for q-PCR. By gold standard, 39 of the dyspeptic patients (51.3%) were found to be infected. Conclusions: With the identified cutoff value, the q-PCR assay diagnosed H. pylori infection with an accuracy slightly superior to that of RUT. However, the possibility that low counts detected only by q-PCR represent true infections warrants further investigation. Normalization of bacterial counts for standardization of q-PCR H. pylori assays is recommended.
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Keywords: culture, Helicobacter pylori, normalization, rapid urease test, quantitative PCR
INTRODUCTION Helicobacter pylori is a Gram-negative microaerophilic, curved bacterium. It is one of the most prevalent human pathogens infecting the stomachs of more than half of the world’s population.1 Its prevalence in developing countries is particularly high with an average of 80% compared to that of 30% in developed countries.2 However, there is significant variation in its prevalence from one country to another in the same region.1 In the Middle East for example, Saudi Arabia has the lowest prevalence (, 50%) while Egypt has the highest (80%).3 H. pylori has been subject to intense research since it was established as a risk factor for chronic active gastritis, peptic ulcer, gastric adenocarcinoma, and gastric lymphoma.4,5 Various tests have been developed for the diagnosis of infection with H. pylori and/or confirmation of its eradication. Non-invasive tests include serology, urea breath test and antigen stool assay while invasive tests comprise histological detection, culture, rapid urease test (RUT) and polymerase chain reaction (PCR).6 The choice among these tests depends on the cost and availability, clinical setting, prevalence of H. pylori infection in the population and, most importantly, the performance of the test itself. So far, RUT seems to have the highest sensitivity and specificity as well as positive predictive value1 and is increasingly being used as a reference.7 However, until today there is no consensus gold standard for the diagnosis of H. pylori infection. The last decade witnessed an increasing interest in the use of quantitative PCR (q-PCR) in the detection of H. pylori in clinical samples.8 Studies have shown the technique to be particularly advantageous with certain types of samples, such as paraffin-embedded formalin-fixed specimens9 and biopsies obtained from patients during episodes of peptic ulcer bleeding10. It also has been found to be useful for assessment of bacterial density in the mucosa.9 In addition, q-PCR has been employed for simultaneous detection of both the bacterium as well as its resistance or virulence genes.11,12 However, there have been significant differences among the described assays in terms of design, detection limit and gold standard used for comparison.9,12 – 15 Furthermore, there has
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Table 1. Demographic and clinical characteristics of patients included in the study (N ¼ 76).
Gender M:F Age (mean ^ SD) Endoscopic finding* Gastritis Duodenitis Esophagitis Gastric ulcer Duodenal ulcer Esophageal ulcer
39:37 35.5 ^ 13.8 No. % 61 80.2 8 10.5 7 9.2 5 6.6 10 13.1 1 1.3
* Some patients had more than one endoscopic finding
been no attempt to normalize bacterial counts to enable reliable comparison of results between studies. Q-PCR assays for the diagnosis of H. pylori infection remain, therefore, largely unstandardized.1 The purpose of the current study was to describe a normalized q-PCR assay for comparable quantification of H. pylori in fresh biopsies from dyspeptic patients, and assess its accuracy in establishing diagnosis of the infection.
SUBJECTS AND METHODS Study subjects and biopsy collection Seventy-six subjects were recruited from among patients scheduled for upper gastrointestinal tract endoscopy at the University of Science and Technology Hospital, Sana’a, Yemen. Endoscopy and biopsy collection were performed under sedation by gastroenterologists using sterile forceps; three antral gastric mucosal biopsy specimens (approximately 2 cm) were obtained from each patient. The clinical features of the recruited patients is presented in Table 1. The study was approved by the Higher Education Council at Sana’a University; verbal consent was obtained from the patients to include their samples in the study.
Detection of H. pylori by rapid urease test (RUT) One specimen from each subject was immediately assayed for the urease enzyme of H. pylori using the Pronto Dry test16 according to the manufacturer’s instructions (Medical Instrument Corporation, Solothurn, Switzerland). The principle of the test is based on hydrolysis of urea in the supplied medium by bacterial urease into carbon dioxide and ammonia,
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causing a rise in the pH that is detected colorimetrically.
40
Detection of H. pylori by culture
Detection of H. pylori by Q-PCR The third biopsy specimen was placed in 1 ml sterile normal saline and stored at -258C. Upon analysis, the specimen was thawed and a 25 mg, finely cut piece of it was used for DNA extraction using the PureLink Genomic DNA kit according to the manufacturer’s instructions (Life Technologies, USA). DNA was eluted in 100 ml elution buffer. A commercial TaqMan q-PCR assay (Primerdesign, UK) was used to quantify actin-beta (ACTB) gene copies in the extracts according to the manufacturer’s instructions. This was done to validate the extraction and also to normalize bacterial counts as described below. Extracts showing inhibition in this assay or a threshold cycle (Ct) higher than 30 were not considered for further testing (low DNA yield) and extraction was redone. For detection and quantification of H. pylori in the DNA extracts, an optimized, ready to use TaqMan q-PCR kit (Primerdesign, UK) was used. The kit included ureA gene-specific primers and FAM-labeled probes with propriety sequences, and a plasmidbased positive control for standard curve construction. Q-PCR reaction was carried out in total volume of 20 ml:10 ml Precision MasterMix (Primerdesign, UK), 1 ml primer/probe mix, 4 ml water, and 5 ml DNA sample. The reactions were run on an ABI 7000 real-time PCR platform (Applied Biosystems, USA) using the program described by the manufacturer.
36
32
Ct
The second gastric biopsy specimen from each subject was immediately placed in 1 ml Tryptone Soy Broth containing 20% glycerol and stored at -808C until cultured.17 Before culturing, the biopsy was thawed at room temperature, homogenized (Omni, USA), and 200 ml were inoculated on Columbia Blood Agar supplemented with Skirrow antibiotics supplement (5.0 mg vancomycin, 2.5 mg trimethoprim, and 1250 IU Polymyxin B), 5% human blood, and 5 ml horse serum.18 The inoculated plates were incubated in an anaerobic jar at 378C for ten days under microaerophillic conditions created by using the CampyGen kit (Oxoid, England). Gram-negative bacteria forming tiny, translucent grey colonies that were urease-, catalase- and oxidase-positive, were identified as H. pylori.19
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24
20
16
1
2
3
4
5
6
Log C0 Detector: FAM, Slope: –3.958674, Intercept: 41.623753, R2: 0.997584
Figure 1. Standard curve of the H. pylori TaqMan q-PCR assay. Serial dilutions of plasmid-based positive control were prepared with final concentrations of 10-106 copies/reaction. Assay was run as described in the text. The curve was obtained by plotting log DNA copies count against threshold cycle (Ct) values.
Bacterial counts in copies/reaction were calculated using the obtained standard curve (Figure 1); a sample was considered positive if it resulted in an amplification plot with Ct #40; i.e., 5 copies/reaction (equivalent to 4 bacteria per mg of the biopsy). Bacterial density were calculated by normalizing absolute counts to human ACTB gene copy number, and expressed as number of bacteria per 100 human cells, given there are two ACTB gene copies per human cell.
Statistical analysis Data were summarized as means and standard deviations (SD) or percentages as appropriate. A patient was considered to be infected with H. pylori when two or more assays were positive (gold standard). Because q-PCR resulted in a high rate of false positives, a receiver operating characteristic (ROC) curve analysis was performed to determine
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had low bacterial counts, the cutoff value was recalculated using ROC curve analysis of the normalized counts (bacterial density) (Figure 2). Based on the curve coordinates obtained, a cutoff value of 1 bacteria per 100 human cells was chosen to maintain the highest sensitivity and specificity (see below).
ROC Curve
1.0
0.8
Sensitivity
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0.6
Ultimate performance of the three assays 0.4
0.2
0.0 0.0
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0.4
0.6
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Figure 2. Receiver operating characteristic curve for the normalized q-PCR assay. The area under the curve is 0.98.
normalized q-PCR cut-off values that demonstrated the best sensitivity and specificity. Sensitivity, specificity, positive predictive-value (PPV) and negative predictive-value (NPV), as percentages and 95% confidence intervals, for each of the three assays against the defined gold standard were then calculated. Agreement among the three tests was assessed using Cohen’s kappa statistic, while significance of differences in sensitivity and specificity were sought using McNemar’s test. All statistical analyses was performed using the IBM Statistical Package for the Social Sciences (SPSS) software version 20.
RESULTS Detection of H. pylori by the three assays H. pylori was detected in 32 patients (42.1%) by culture, and in 40 (52.6%) by RUT. Using the initial cutoff value for q-PCR (Ct #40), 60 patients (78.9%) tested positive. Bacterial density as determined by the later assay normalized to human DNA was in the range of 0.005 to 486 bacteria per 100 human cells (mean of 29). According to the gold standard definition ($2 positive tests), 41 patients (53.9%) were initially considered as infected.
Recalculating the cutoff value for q-PCR Compared to the gold standard, the q-PCR assay showed low specificity (45.7%) with 19 falsepositive results. Because most of these false-positives
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The number of dyspeptic patients finally diagnosed with H. pylori infection according to the gold standard was 39; i.e., a prevalence of 51.3%. The performance of the three assays against this is presented in Table 2. Culture showed the highest specificity (97.3%) but the lowest sensitivity (79.5%). With the new cutoff value, 17 of the 19 initially false-positives by q-PCR became negative, dramatically increasing specificity of q-PCR to 94.9%; however, two of the positive cases by gold standard tested negative dropping initially 100% sensitivity to 94.6%. RUT showed an identical sensitivity to that of q-PCR but slightly lower specificity. Overall accuracy was 94.7%, 93.4% and 90.7% for q-PCR, RUT and culture, respectively. RUT and q-PCR showed the highest agreement (kappa ¼ 0.736), followed by q-PCR and culture (kappa ¼ 0.712), and finally RUT and culture (kappa ¼ 0.635). Differences in sensitivity and specificity were not found to be statistically significant.
DISCUSSION A major aspect of strength in the current study was the use of normalized rather than absolute counts to measure bacterial load in the samples. Due to variations in the amount of biopsy used for DNA extraction as well as in the efficiency of extraction itself, comparison of bacterial loads between samples, even within the same study, using absolute counts is obviously not reliable. The importance of this has so far been highlighted only by Kobayashi et al., and Riberio et al., who normalized the counts of H. pylori to human DNA in picograms/nanograms.9,15. Another aspect of standardization in the current study was that extracts showing Ct .30 in the ACTB gene assays where not considered for further testing and DNA extraction was redone. That ensured exclusion of extracts with low DNA yield or/and PCR inhibitors. Several real-time PCR assays for the detection of H. pylori have been described in the literature. Different genes have been used as targets in these assays including the ureC, ureA, 16S rRNA and 23S
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Table 2. Sensitivity, specificity, PPV and NPV (as percentages and 95% confidence intervals) for each of the three assays used for detection of H. pylori in the biopsies.
Assay
Sensitivity*
Specificity*
PPV
NPV
Culture RUT Q-PCR
79.5 (66.7-92.3) 94.9 (87.2-100) 94.9 (87.2-100)
97.3 (91.9-100) 91.9 (81.1-100) 94.6 (86.5-100)
96.9 (90.6-100) 92.5 (82.5-100) 94.9 (87.2-100)
81.8 (68.2-93.2) 94.4 (86.1-100) 94.6 (86.5-100)
* Differences in sensitivity and specificity were not found to be statistically significant.
rRNA genes.9,12 – 14 The commercial kit used in the current study targets the ureA gene, which is present as a single copy per bacterial genome. However, what is probably most critical to the performance of PCR is the primers design rather than the target gene selected.20 The detection limit of the assay was set at five copies per reaction, which is equivalent to four bacterial DNA per mg sample. This is consistent with the assay described by Schabereiter-Gurtner et al.,12 in which a detection limit of 2.2 bacteria per PCR reaction and 5-8 bacteria per mg stool was reported. Lascols et al.,14 reported a higher detection limit at 30 bacteria per PCR reaction while He et al.,13 were only able to accurately quantify down to 1000 bacteria per reaction, although 10-100 bacterial DNA was qualitatively detectable. Obviously, without reporting detection limits as the number of bacteria per unit sample weight or volume, comparison between studies becomes difficult. Regardless of the differences in assay design, the reported sensitivities and specificities for q-PCR for diagnosis of H. pylori have been high, being in the range of 97-100% for sensitivity, and 94.6-100% for specificity.9,12,14 The sensitivity in the current study was 100% initially and dropped to 94.9% after recalculating the diagnostic q-PCR cutoff value, which is still close to the reported range. The specificity, however, was initially very low (45.7%). The high number of false positive cases with q-PCR was probably because the bacterial load in these cases was too low to be detectable by RUTor culture. In the study by Lascols et al.,14 nine of the cases that were negative by both culture and histology tested positive by q-PCR. That was also attributed to a low bacterial count in the samples; however, the specificity of the assay was not significantly compromised because additional non-invasive tests were included and confirmed the diagnosis in four out of the nine cases. Similarly, He et al.,13 detected H. pylori DNA by q-PCR in 24 samples out of 27 that were culture negative.
Unlike the current study, however, the bacterial load in those samples was very high, which is difficult to explain. The ability of q-PCR to detect bacteria at very low counts can compromise its specificity when other techniques such as culture, RUT or/and histology are used as gold standard. This becomes particularly evident when the proportion of study subjects harboring low counts is high, which was probably the case in the current study. To improve specificity of the assay, ROC curve analysis of normalized q-PCR counts was, therefore, performed. The identified cutoff value (1 bacteria per 100 human cells) dramatically improved the specificity, although sensitivity was slightly affected. A similar approach was used previously7 to calculate the delta cutoff value for urease breath test. One possibility remains, however, that low bacterial counts detected only by q-PCR do represent true infections. This possibility is strongly supported by findings from the study of Ribeiro et al., in which H. pylori was detected by q-PCR in 16 out of 81 (19.7%) gastric biopsies that had previously tested negative by reference methods.15 Indeed, a significant proportion (20-50%) of dyspeptic patients are usually not diagnosed with H. pylori infection, which may be attributed to low sensitivity of the routinely used assays rather than actual absence of infection. As such, q-PCR may ultimately prove to be the most accurate method for diagnosis of H. pylori infection and replace currently used tests. One limitation to the current study was that the specimens for culture were stored at -808C until all of them were processed together. While this has been previously described in the literature,17 it may have probably reduced the sensitivity of the assay. Another limitation was that a proper history about recent antibiotic intake was not obtained with the assumption that patients admitted for diagnosis
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rather than follow up, were unlikely to have received previous H. pylori eradication therapy. In other words, recent antibiotic use in some of the patients was not totally excluded. However, any such history may have only influenced the overall detection rate (prevalence) but not the comparisons among the assays.
CONCLUSION In conclusion, with the identified cutoff value, the q-PCR assay diagnosed H. pylori infection with an accuracy slightly superior to that of RUT. However, if low counts detected only by q-PCR are considered as true infection, the assay will have much higher sensitivity than RUT, a scenario that warrants further
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investigation. In any case, normalization of bacterial counts is probably an important aspect of standardization of q-PCR H. pylori assays since it enables reliable comparison of results among studies.
ACKNOWLEDGEMENTS The research work was funded by Al-Saeed foundation for Science and Culture. All laboratory work was performed at the Molecular Research Laboratory, University of Science and Technology, Yemen. We would like to thank Dr. Mohammed Abdul-Kader Al-Bughali for his assistance with the laboratory work.
REFERENCES 1. Hunt RH, Xiao SD, Megraud F, Leon-Barua R, Bazzoli F, van der Merwe S, Vaz Coelho LG, Fock M, Fedail S, Cohen H, Malfertheiner P, Vakil N, Hamid S, Goh KL, Wong BC, Krabshuis J, Le Mair A, World Gastroenterology Organization. Helicobacter pylori in developing countries. World Gastroenterology Organisation Global Guideline. J Gastrointestin Liver Dis. 2011 Sep;20(3):299 –304. 2. Bardhan PK. Epidemiological features of Helicobacter pylori infection in developing countries. Clin Infect Dis. 1997 Nov;25(5):973 – 978. 3. Khedmat H, Karbasi-Afshar R, Agah S, Taheri S. Helicobacter pylori Infection in the general population: A Middle Eastern perspective. Caspian J Intern Med. 2013 Fall;4(4):745 –753. 4. Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984 Jun 16;1(8390): 1311–1315. 5. Peter S, Beglinger C. Helicobacter pylori and gastric cancer: the causal relationship. Digestion. 2007;75(1):25– 35. 6. Monteiro L, de Mascarel A, Sarrasqueta AM, Bergey B, Barberis C, Talby P, Roux D, Shouler L, Goldfain D, Lamouliatte H, Mégraudgraud F. Diagnosis of Helicobacter pylori infection: noninvasive methods compared to invasive methods and evaluation of two new tests. Am J Gastroenterol. 2001 Feb;96(2): 353 – 358. 7. Calvet X, Sanchez-Delgado J, Montserrat A, Lario S, Ramirez-Lazaro MJ, Quesada M, Casalots A, Suárez D, Campo R, Brullet E, Junquera F, Sanfeliu I, Segura F. Accuracy of diagnostic tests for Helicobacter pylori: a reappraisal. Clin Infect Dis. 2009 May 15; 48(10):1385– 1391.
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8. Megraud F, Lehours P. Helicobacter pylori detection and antimicrobial susceptibility testing. Clin Microbiol Rev. 2007 Apr;20(2):280 –322. 9. Kobayashi D, Eishi Y, Ohkusa T, Ishige, Suzuki T, Minami J, Yamada T, Takizawa T, Koike M. Gastric mucosal density of Helicobacter pylori estimated by real-time PCR compared with results of urea breath test and histological grading. Med Microbiol. 2002 Apr;51(4):305 –311. 10. Ramirez-Lazaro MJ, Lario S, Casalots A, Sanfeliu E, Boix L, Garcia-Iglesias P, Sánchez-Delgado J, Montserrat A, Bella-Cueto MR, Gallach M, Sanfeliu I, Segura F, Calvet X. Real-time PCR improves Helicobacter pylori detection in patients with peptic ulcer bleeding. PLoS One. 2011;6(5):e20009. 11. Karaman M, Abacıog˘lu H, Topalak OS, Sims ek I. Molecular detection of Helicobacter pylori vacA and cagA genes in gastric tissue specimens of patients with peptic ulcer disease and non-ulcer dyspepsia. Mikrobiyoloji bülteni. 2011;45(1):11. 12. Schabereiter-Gurtner C, Hirschl AM, Dragosics B, Hufnagl P, Puz S, Kovách Z, Rotter M, Makristathis A. Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy specimens. J Clin Microbiol. 2004;42(10): 4512 –4518. 13. He Q, Wang J-P, Osato M, Lachman LB. Real-time quantitative PCR for detection of Helicobacter pylori. J clin microbiol. 2002;40(10): 3720 – 3728. 14. Lascols C, Lamarque D, Costa JM, Copie-Bergman C, Le Glaunec JM, Deforges L, Soussy CJ, Petit JC, Delchier JC, Tankovic J. Fast and accurate quantitative detection of Helicobacter pylori and identification of clarithromycin resistance mutations
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in H. pylori isolates from gastric biopsy specimens by real-time PCR. J Clin Microbiol. 2003 Oct;41(10): 4573 –4577.
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method. East Mediterr Health J. 2008;14(2): 268 –275.
15. Ribeiro ML, Ecclissato CC, Mattos RG, Mendonca S, Pedrazzoli J Jr. Quantitative real-time PCR for the clinical detection of Helicobacter pylori. Genet Mol Biol. 2007;30(2):431 –434.
18. Pandy HB, Patel JS, Sodagar NR, Singh NR, Salvi PN, Patel SB, Naik KB, Agravat HH. Comparison of invasive tests with serology to diagnosed Helicobacter pylori infections in symptomatic patients. J Cell Tissue Res. 2009;9(3):2019 –2022.
16. Said RM, Cheah PL, Chin SC, Goh KL. Evaluation of a new biopsy urease test: Pronto Dry, for the diagnosis of Helicobacter pylori infection. Eur J Gastroenterol Hepatol. 2004 Feb;16(2):195 –199.
19. Kumar A, Imran M, Singh AK, Chandel D, Talwar A. Detection of Helicobacter pylori in gastroduodenal diseases by Real-time PCR. J Biomed Sci and Res. 2010;2(3):170–178.
17. Al-Sulami A, Al-Kiat H, Bakker L, Hunoon H. Primary isolation and detection of Helicobacter pylori from dyspeptic patients: a simple, rapid
20. Yamamoto Y. PCR in diagnosis of infection: detection of bacteria in cerebrospinal fluids. Clin Diagn Lab Immunol. 2002 May;9(3):508 –514.
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Normalized real-time PCR for diagnosis of H. pylori infection Ebtisam E. Al-Moayad1, Saeed M. Alghalibi1, Hassan A. Al-Shamahy2, Akram T. Nasher3, Nezar N. Al-hebshi4,5 ABSTRACT Address for Correspondence: Nezar N. Al-hebshi 1 Department of Biology, Microbiology Section, Faculty of Science, Sana’a University, Yemen 2 Department of Medical Microbiology, Faculty of Medicine and Health Sciences, Sana’a University, Yemen 3 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Sana’a University, Yemen 4 Molecular Biology Research Laboratory, Faculty of Medical Sciences, University of Science and Technology, Sana’a, Yemen 5 Medical Research Center, Jazan University, Jazan, Kingdom of Saudi Arabia Email: [email protected]
http://dx.doi.org/10.5339/qmj.2014.19 Submitted: 16 October 2014 Accepted: 26 November 2014 ª 2014 Al-Moayad, Alghalibi, Al-Shamahy, Nasher, Al-hebshi, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 4.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.
Cite this article as: Al-Moayad EE, Alghalibi SM, Al-Shamahy HA, Nasher AT, Al-hebshi NN. Normalized real-time PCR for diagnosis of H. pylori infection, Qatar Medical Journal 2014:19 http://dx.doi.org/10.5339/ qmj.2014.19
Objectives: There is increasing interest in the use of quantitative PCR (q-PCR) for diagnosis of H. pylori infection. However, the assay remains largely unstandardized, making comparison between studies unreliable. The objective of this study was to assess accuracy of a normalized q-PCR assay for diagnosis of the infection. Subjects and methods: Seventy-six fresh gastric biopsy specimens were collected from patients undergoing upper gastrointestinal tract endoscopy and examined by rapid urease test (RUT), culture, and a commercial TaqMan q-PCR assay targeting the ureA gene. Counts obtained from the latter assay were normalized to the human ACTB gene. A subject was considered to be infected if two or more assays were positive. Results: The detection rates were 42.1%, 52.6%, and 78.9% by culture, RUT and q-PCR, respectively. Bacterial density ranged 0.005 to 4800 bacteria per 100 human cells. Because q-PCR showed low initial specificity (45.7%), the cutoff value for the assay was recalculated as 1 bacterium per 100 human cells, using ROC curve analysis. Accordingly, the sensitivities and specificities were 79.5% and 97.3%, respectively, for culture; 94.9% and 91.9%, respectively, for RUT; and 94.9% and 94.6%, respectively, for q-PCR. By gold standard, 39 of the dyspeptic patients (51.3%) were found to be infected. Conclusions: With the identified cutoff value, the q-PCR assay diagnosed H. pylori infection with an accuracy slightly superior to that of RUT. However, the possibility that low counts detected only by q-PCR represent true infections warrants further investigation. Normalization of bacterial counts for standardization of q-PCR H. pylori assays is recommended.
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Keywords: culture, Helicobacter pylori, normalization, rapid urease test, quantitative PCR
INTRODUCTION Helicobacter pylori is a Gram-negative microaerophilic, curved bacterium. It is one of the most prevalent human pathogens infecting the stomachs of more than half of the world’s population.1 Its prevalence in developing countries is particularly high with an average of 80% compared to that of 30% in developed countries.2 However, there is significant variation in its prevalence from one country to another in the same region.1 In the Middle East for example, Saudi Arabia has the lowest prevalence (, 50%) while Egypt has the highest (80%).3 H. pylori has been subject to intense research since it was established as a risk factor for chronic active gastritis, peptic ulcer, gastric adenocarcinoma, and gastric lymphoma.4,5 Various tests have been developed for the diagnosis of infection with H. pylori and/or confirmation of its eradication. Non-invasive tests include serology, urea breath test and antigen stool assay while invasive tests comprise histological detection, culture, rapid urease test (RUT) and polymerase chain reaction (PCR).6 The choice among these tests depends on the cost and availability, clinical setting, prevalence of H. pylori infection in the population and, most importantly, the performance of the test itself. So far, RUT seems to have the highest sensitivity and specificity as well as positive predictive value1 and is increasingly being used as a reference.7 However, until today there is no consensus gold standard for the diagnosis of H. pylori infection. The last decade witnessed an increasing interest in the use of quantitative PCR (q-PCR) in the detection of H. pylori in clinical samples.8 Studies have shown the technique to be particularly advantageous with certain types of samples, such as paraffin-embedded formalin-fixed specimens9 and biopsies obtained from patients during episodes of peptic ulcer bleeding10. It also has been found to be useful for assessment of bacterial density in the mucosa.9 In addition, q-PCR has been employed for simultaneous detection of both the bacterium as well as its resistance or virulence genes.11,12 However, there have been significant differences among the described assays in terms of design, detection limit and gold standard used for comparison.9,12 – 15 Furthermore, there has
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Table 1. Demographic and clinical characteristics of patients included in the study (N ¼ 76).
Gender M:F Age (mean ^ SD) Endoscopic finding* Gastritis Duodenitis Esophagitis Gastric ulcer Duodenal ulcer Esophageal ulcer
39:37 35.5 ^ 13.8 No. % 61 80.2 8 10.5 7 9.2 5 6.6 10 13.1 1 1.3
* Some patients had more than one endoscopic finding
been no attempt to normalize bacterial counts to enable reliable comparison of results between studies. Q-PCR assays for the diagnosis of H. pylori infection remain, therefore, largely unstandardized.1 The purpose of the current study was to describe a normalized q-PCR assay for comparable quantification of H. pylori in fresh biopsies from dyspeptic patients, and assess its accuracy in establishing diagnosis of the infection.
SUBJECTS AND METHODS Study subjects and biopsy collection Seventy-six subjects were recruited from among patients scheduled for upper gastrointestinal tract endoscopy at the University of Science and Technology Hospital, Sana’a, Yemen. Endoscopy and biopsy collection were performed under sedation by gastroenterologists using sterile forceps; three antral gastric mucosal biopsy specimens (approximately 2 cm) were obtained from each patient. The clinical features of the recruited patients is presented in Table 1. The study was approved by the Higher Education Council at Sana’a University; verbal consent was obtained from the patients to include their samples in the study.
Detection of H. pylori by rapid urease test (RUT) One specimen from each subject was immediately assayed for the urease enzyme of H. pylori using the Pronto Dry test16 according to the manufacturer’s instructions (Medical Instrument Corporation, Solothurn, Switzerland). The principle of the test is based on hydrolysis of urea in the supplied medium by bacterial urease into carbon dioxide and ammonia,
Normalized real-time PCR for diagnosis of H. pylori infection
Al-Moayad et al.
causing a rise in the pH that is detected colorimetrically.
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Detection of H. pylori by culture
Detection of H. pylori by Q-PCR The third biopsy specimen was placed in 1 ml sterile normal saline and stored at -258C. Upon analysis, the specimen was thawed and a 25 mg, finely cut piece of it was used for DNA extraction using the PureLink Genomic DNA kit according to the manufacturer’s instructions (Life Technologies, USA). DNA was eluted in 100 ml elution buffer. A commercial TaqMan q-PCR assay (Primerdesign, UK) was used to quantify actin-beta (ACTB) gene copies in the extracts according to the manufacturer’s instructions. This was done to validate the extraction and also to normalize bacterial counts as described below. Extracts showing inhibition in this assay or a threshold cycle (Ct) higher than 30 were not considered for further testing (low DNA yield) and extraction was redone. For detection and quantification of H. pylori in the DNA extracts, an optimized, ready to use TaqMan q-PCR kit (Primerdesign, UK) was used. The kit included ureA gene-specific primers and FAM-labeled probes with propriety sequences, and a plasmidbased positive control for standard curve construction. Q-PCR reaction was carried out in total volume of 20 ml:10 ml Precision MasterMix (Primerdesign, UK), 1 ml primer/probe mix, 4 ml water, and 5 ml DNA sample. The reactions were run on an ABI 7000 real-time PCR platform (Applied Biosystems, USA) using the program described by the manufacturer.
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Ct
The second gastric biopsy specimen from each subject was immediately placed in 1 ml Tryptone Soy Broth containing 20% glycerol and stored at -808C until cultured.17 Before culturing, the biopsy was thawed at room temperature, homogenized (Omni, USA), and 200 ml were inoculated on Columbia Blood Agar supplemented with Skirrow antibiotics supplement (5.0 mg vancomycin, 2.5 mg trimethoprim, and 1250 IU Polymyxin B), 5% human blood, and 5 ml horse serum.18 The inoculated plates were incubated in an anaerobic jar at 378C for ten days under microaerophillic conditions created by using the CampyGen kit (Oxoid, England). Gram-negative bacteria forming tiny, translucent grey colonies that were urease-, catalase- and oxidase-positive, were identified as H. pylori.19
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16
1
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6
Log C0 Detector: FAM, Slope: –3.958674, Intercept: 41.623753, R2: 0.997584
Figure 1. Standard curve of the H. pylori TaqMan q-PCR assay. Serial dilutions of plasmid-based positive control were prepared with final concentrations of 10-106 copies/reaction. Assay was run as described in the text. The curve was obtained by plotting log DNA copies count against threshold cycle (Ct) values.
Bacterial counts in copies/reaction were calculated using the obtained standard curve (Figure 1); a sample was considered positive if it resulted in an amplification plot with Ct #40; i.e., 5 copies/reaction (equivalent to 4 bacteria per mg of the biopsy). Bacterial density were calculated by normalizing absolute counts to human ACTB gene copy number, and expressed as number of bacteria per 100 human cells, given there are two ACTB gene copies per human cell.
Statistical analysis Data were summarized as means and standard deviations (SD) or percentages as appropriate. A patient was considered to be infected with H. pylori when two or more assays were positive (gold standard). Because q-PCR resulted in a high rate of false positives, a receiver operating characteristic (ROC) curve analysis was performed to determine
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had low bacterial counts, the cutoff value was recalculated using ROC curve analysis of the normalized counts (bacterial density) (Figure 2). Based on the curve coordinates obtained, a cutoff value of 1 bacteria per 100 human cells was chosen to maintain the highest sensitivity and specificity (see below).
ROC Curve
1.0
0.8
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Ultimate performance of the three assays 0.4
0.2
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0.4
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Figure 2. Receiver operating characteristic curve for the normalized q-PCR assay. The area under the curve is 0.98.
normalized q-PCR cut-off values that demonstrated the best sensitivity and specificity. Sensitivity, specificity, positive predictive-value (PPV) and negative predictive-value (NPV), as percentages and 95% confidence intervals, for each of the three assays against the defined gold standard were then calculated. Agreement among the three tests was assessed using Cohen’s kappa statistic, while significance of differences in sensitivity and specificity were sought using McNemar’s test. All statistical analyses was performed using the IBM Statistical Package for the Social Sciences (SPSS) software version 20.
RESULTS Detection of H. pylori by the three assays H. pylori was detected in 32 patients (42.1%) by culture, and in 40 (52.6%) by RUT. Using the initial cutoff value for q-PCR (Ct #40), 60 patients (78.9%) tested positive. Bacterial density as determined by the later assay normalized to human DNA was in the range of 0.005 to 486 bacteria per 100 human cells (mean of 29). According to the gold standard definition ($2 positive tests), 41 patients (53.9%) were initially considered as infected.
Recalculating the cutoff value for q-PCR Compared to the gold standard, the q-PCR assay showed low specificity (45.7%) with 19 falsepositive results. Because most of these false-positives
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The number of dyspeptic patients finally diagnosed with H. pylori infection according to the gold standard was 39; i.e., a prevalence of 51.3%. The performance of the three assays against this is presented in Table 2. Culture showed the highest specificity (97.3%) but the lowest sensitivity (79.5%). With the new cutoff value, 17 of the 19 initially false-positives by q-PCR became negative, dramatically increasing specificity of q-PCR to 94.9%; however, two of the positive cases by gold standard tested negative dropping initially 100% sensitivity to 94.6%. RUT showed an identical sensitivity to that of q-PCR but slightly lower specificity. Overall accuracy was 94.7%, 93.4% and 90.7% for q-PCR, RUT and culture, respectively. RUT and q-PCR showed the highest agreement (kappa ¼ 0.736), followed by q-PCR and culture (kappa ¼ 0.712), and finally RUT and culture (kappa ¼ 0.635). Differences in sensitivity and specificity were not found to be statistically significant.
DISCUSSION A major aspect of strength in the current study was the use of normalized rather than absolute counts to measure bacterial load in the samples. Due to variations in the amount of biopsy used for DNA extraction as well as in the efficiency of extraction itself, comparison of bacterial loads between samples, even within the same study, using absolute counts is obviously not reliable. The importance of this has so far been highlighted only by Kobayashi et al., and Riberio et al., who normalized the counts of H. pylori to human DNA in picograms/nanograms.9,15. Another aspect of standardization in the current study was that extracts showing Ct .30 in the ACTB gene assays where not considered for further testing and DNA extraction was redone. That ensured exclusion of extracts with low DNA yield or/and PCR inhibitors. Several real-time PCR assays for the detection of H. pylori have been described in the literature. Different genes have been used as targets in these assays including the ureC, ureA, 16S rRNA and 23S
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Table 2. Sensitivity, specificity, PPV and NPV (as percentages and 95% confidence intervals) for each of the three assays used for detection of H. pylori in the biopsies.
Assay
Sensitivity*
Specificity*
PPV
NPV
Culture RUT Q-PCR
79.5 (66.7-92.3) 94.9 (87.2-100) 94.9 (87.2-100)
97.3 (91.9-100) 91.9 (81.1-100) 94.6 (86.5-100)
96.9 (90.6-100) 92.5 (82.5-100) 94.9 (87.2-100)
81.8 (68.2-93.2) 94.4 (86.1-100) 94.6 (86.5-100)
* Differences in sensitivity and specificity were not found to be statistically significant.
rRNA genes.9,12 – 14 The commercial kit used in the current study targets the ureA gene, which is present as a single copy per bacterial genome. However, what is probably most critical to the performance of PCR is the primers design rather than the target gene selected.20 The detection limit of the assay was set at five copies per reaction, which is equivalent to four bacterial DNA per mg sample. This is consistent with the assay described by Schabereiter-Gurtner et al.,12 in which a detection limit of 2.2 bacteria per PCR reaction and 5-8 bacteria per mg stool was reported. Lascols et al.,14 reported a higher detection limit at 30 bacteria per PCR reaction while He et al.,13 were only able to accurately quantify down to 1000 bacteria per reaction, although 10-100 bacterial DNA was qualitatively detectable. Obviously, without reporting detection limits as the number of bacteria per unit sample weight or volume, comparison between studies becomes difficult. Regardless of the differences in assay design, the reported sensitivities and specificities for q-PCR for diagnosis of H. pylori have been high, being in the range of 97-100% for sensitivity, and 94.6-100% for specificity.9,12,14 The sensitivity in the current study was 100% initially and dropped to 94.9% after recalculating the diagnostic q-PCR cutoff value, which is still close to the reported range. The specificity, however, was initially very low (45.7%). The high number of false positive cases with q-PCR was probably because the bacterial load in these cases was too low to be detectable by RUTor culture. In the study by Lascols et al.,14 nine of the cases that were negative by both culture and histology tested positive by q-PCR. That was also attributed to a low bacterial count in the samples; however, the specificity of the assay was not significantly compromised because additional non-invasive tests were included and confirmed the diagnosis in four out of the nine cases. Similarly, He et al.,13 detected H. pylori DNA by q-PCR in 24 samples out of 27 that were culture negative.
Unlike the current study, however, the bacterial load in those samples was very high, which is difficult to explain. The ability of q-PCR to detect bacteria at very low counts can compromise its specificity when other techniques such as culture, RUT or/and histology are used as gold standard. This becomes particularly evident when the proportion of study subjects harboring low counts is high, which was probably the case in the current study. To improve specificity of the assay, ROC curve analysis of normalized q-PCR counts was, therefore, performed. The identified cutoff value (1 bacteria per 100 human cells) dramatically improved the specificity, although sensitivity was slightly affected. A similar approach was used previously7 to calculate the delta cutoff value for urease breath test. One possibility remains, however, that low bacterial counts detected only by q-PCR do represent true infections. This possibility is strongly supported by findings from the study of Ribeiro et al., in which H. pylori was detected by q-PCR in 16 out of 81 (19.7%) gastric biopsies that had previously tested negative by reference methods.15 Indeed, a significant proportion (20-50%) of dyspeptic patients are usually not diagnosed with H. pylori infection, which may be attributed to low sensitivity of the routinely used assays rather than actual absence of infection. As such, q-PCR may ultimately prove to be the most accurate method for diagnosis of H. pylori infection and replace currently used tests. One limitation to the current study was that the specimens for culture were stored at -808C until all of them were processed together. While this has been previously described in the literature,17 it may have probably reduced the sensitivity of the assay. Another limitation was that a proper history about recent antibiotic intake was not obtained with the assumption that patients admitted for diagnosis
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rather than follow up, were unlikely to have received previous H. pylori eradication therapy. In other words, recent antibiotic use in some of the patients was not totally excluded. However, any such history may have only influenced the overall detection rate (prevalence) but not the comparisons among the assays.
CONCLUSION In conclusion, with the identified cutoff value, the q-PCR assay diagnosed H. pylori infection with an accuracy slightly superior to that of RUT. However, if low counts detected only by q-PCR are considered as true infection, the assay will have much higher sensitivity than RUT, a scenario that warrants further
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investigation. In any case, normalization of bacterial counts is probably an important aspect of standardization of q-PCR H. pylori assays since it enables reliable comparison of results among studies.
ACKNOWLEDGEMENTS The research work was funded by Al-Saeed foundation for Science and Culture. All laboratory work was performed at the Molecular Research Laboratory, University of Science and Technology, Yemen. We would like to thank Dr. Mohammed Abdul-Kader Al-Bughali for his assistance with the laboratory work.
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